Date of Award

2022

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Physiology

Abstract

Altered kinase activity and localization is gaining appreciation as a mechanism contributing to cardiovascular disease. Previous work suggests that the kinase glycogen synthase kinase 3 β (GSK-3β) regulates contractile function of the myofilament, but this work was performed in vitro. The goal of this work was to investigate GSK-3β’s in vivo role in regulating myofilament function, the mechanisms involved, and the translational relevance in disease. GSK-3β’s myofilament localization was confirmed in wild type mouse and human tissue via western blot of myofilament enriched tissue and immunofluorescence. These experiments also revealed that GSK-3β phosphorylated at Y216 binds to the z-disc. We showed pY216 was necessary and sufficient for z-disc binding using adenoviruses for wildtype (WT) Y216F, and Y216E. We utilized skinned cardiomyocytes from inducible cardiomyocyte-specific GSK-3β KO (KO) mice and found several z-disc proteins as the myofilament phospho-substrates of GSK-3β using mass spectrometry. Functionally, myocytes from the GSK-3β KO animals failed to exhibit calcium sensitization with stretch indicating a loss of Length-Dependent Activation (LDA), the molecular mechanism underlying the Frank Starling Law of the Heart. Titin acts as a length sensor for LDA, and KO mice had decreased titin stiffness compared to Control mice, explaining the lack of LDA. KO mice exhibited no changes in titin isoforms, titin phosphorylation, or other thin filament phosphorylation sites known to affect passive tension or LDA. One of GSK-3β’s z-disc targets, abLIM-1, binds to the z-disc domains of titin that are important for maintaining passive tension. Genetic knockdown of abLIM-1 via siRNA in engineered human heart tissue resulted in enhancement of LDA, indicating abLIM-1 may act as a negative regulator that is modulated by GSK-3β. Lastly, GSK-3β myofilament localization was reduced in LV myocardium from failing human hearts, which correlated with depressed LDA. GSK-3β’s myofilament localization was also explored in a mouse model of myocardial infarction, where myofilament GSK-3β was found to be reduced just 48 hours post injury. We identified a novel mechanism by which GSK-3β localizes to the myofilament to modulate LDA. Importantly, z-disc GSK-3β levels were reduced in heart failure patients, indicating that z-disc localized GSK-3β may be a possible therapeutic target to restore the Frank-Starling mechanism in heart failure patients.

Creative Commons License

Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License.

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